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Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Protocol: ATCCNO: 50655 SPEC: The strain is distributed as a frozen stabilate. See general instructions for thawing and storage of frozen material before proceeding. Remove the frozen ampule from the dry ice and transfer directly to a 35C water bath. After thawing the ampule, transfer the contents to the surface of an ATCC medium 997 or 711 agar plate (20 x 100 mm). Spread the material evenly over the surface of the plate with a sterile spread bar. The food bacterium, Enterobacter aerogenes ATCC 13048, is present in the thawed material. Wrap the plate with time tape or parafilm and incubate upright at 25C. Many trophozoites should be visible within 2-3 days. Transfer the culture every 28 days as follows: Aseptically remove a small block of agar (approximately 5 mm square) containing cysts and, at the edge of an ATCC medium 997 or 711 agar plate containing a lawn of Enterobacter aerogenes ATCC 13048, invert the block onto the surface. Wrap the new plate and incubate as above. The trophozoites will migrate away from the agar block to the opposite edge of the plate. Continue to subculture as above.

Subcultivation

Protocol: ATCCNO: 50655 SPEC: The strain is distributed as a frozen stabilate. See general instructions for thawing and storage of frozen material before proceeding. Remove the frozen ampule from the dry ice and transfer directly to a 35C water bath. After thawing the ampule, transfer the contents to the surface of an ATCC medium 997 or 711 agar plate (20 x 100 mm). Spread the material evenly over the surface of the plate with a sterile spread bar. The food bacterium, Enterobacter aerogenes ATCC 13048, is present in the thawed material. Wrap the plate with time tape or parafilm and incubate upright at 25C. Many trophozoites should be visible within 2-3 days. Transfer the culture every 28 days as follows: Aseptically remove a small block of agar (approximately 5 mm square) containing cysts and, at the edge of an ATCC medium 997 or 711 agar plate containing a lawn of Enterobacter aerogenes ATCC 13048, invert the block onto the surface. Wrap the new plate and incubate as above. The trophozoites will migrate away from the agar block to the opposite edge of the plate. Continue to subculture as above.

Cryopreservation

1. Allow the cells to encyst. To detach cysts from the plate flush the surface with 5 ml fresh ATCC medium 1323 (Page's Balanced Salt Solution). Rub the surface of the plate with a spread bar to detach adhering cysts.

2. Transfer the liquid medium to a sterile centrifuge tube.

3. If the cyst concentration does not exceed 2 x 106 cysts/ml adjust the suspension to that concentration. To adjust the concentration, centrifuge at 600 x g for 5 min and resuspend the pellet in the volume of fresh medium required to yield 2 x 106.

4. While cells are centrifuging prepare a 15% (v/v) solution of sterile DMSO as follows: Add the required volume of DMSO to a glass screw-capped test tube and place it in an ice bath. Allow the DMSO to solidify. Add the required volume of refrigerated medium. Dissolve the DMSO by inverting the tube several times.

*NOTE: If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.

5. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be at least 106 cysts/ml and 7.5% (v/v) DMSO. The equilibration time (the time between addition of DMSO and the start of the cooling cycle) should be no less than 15 min and no longer than 30 min.

7. Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately

-1°C/min.)

8. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen freezer.

9. To establish a culture from the frozen state place an ampule in a water bath set at 35°C (2-3 min). Immerse the vial to a level just above the surface of the frozen material. Do not agitate the vial.

10.Immediately after thawing, aseptically remove the contents of the ampule and distribute to the center of a fresh plate of ATCC medium 711. Distribute the material evenly over the plate using a spread bar. Incubate at 25°C.

Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.